Method for detecting an infection by the hepatitis c virus

ABSTRACT

The invention relates to a method of in-vitro detection of an infection with a hepatitis C virus (HCV) in a biological sample, comprising the simultaneous detection of the HCV capsid protein, and of an antibody directed against said capsid protein, said method using, for capturing the anti-capsid antibodies, a peptide comprising an antigenic fragment derived from the truncated HCV capsid. The invention also relates to the peptide for capturing the anti-capsid antibodies and the kits comprising it.

The invention relates to the in-vitro detection of infection with ahepatitis C virus (HCV), and to the use, for this purpose, of anantigenic peptide derived from the capsid protein of the virus.

TECHNOLOGICAL BACKGROUND OF THE INVENTION

Infection with the hepatitis C virus is a worrying health problem thathas long been recognized, in particular in blood transfusion.

To reduce post-transfusion risks, it is necessary to detect the presenceof the virus itself, before antibodies appear, and as soon as possibleafter contamination. This period between contamination andseroconversion (i.e. the appearance of antibodies) is called the“serological window”.

With a view to having a method that is simple, sensitive, specific,reproducible, inexpensive and easy to use, and can be automated for massscreening for detecting, firstly, the HCV antigen during the serologicalwindow period, and then for monitoring the serological evolution of thepatient after seroconversion, detection of the anti-HCV antibodies,combined with detection of an HCV antigen, has been proposed.

However, this poses a major problem, that of interference between theanti-HCV antibodies present in the serum and labelled anti-HCVantibodies. Thus, the introduction on a solid phase, for the purpose ofdetecting a given antibody, of a target antigen having the same epitopesas those recognized by the labelled antibody or antibodies used forsimultaneous, sandwich detection of an antigen would lead irreversiblyto fixation of labelled antibody/antibodies on the solid phase andtherefore a false positive response of the test.

This is particularly true in a system for simultaneous detection, on thesame solid phase, of anti-HCV capsid antibodies and of HCV capsidantigen. Thus, deposition on the solid phase, for the purpose ofdetecting anti-HCV capsid antibodies, of a capsid antigen that has thesame epitopes as those recognized by the labelled anti-HCV capsidantibody or antibodies used for detecting the capsid antigen leads tofixation of labelled antibody/antibodies on the solid phase and resultsin a false positive response of the test.

To tackle the problem of interference, application EP 1 020 727(Advanced Life Science Institute) proposes a method for simultaneousmeasurement of the HCV capsid antigen and of anti-HCV capsid antibodies(a test of the “Combo” type), in which the antigen is captured andlabelled by antibodies directed against capsid epitopes different fromthe capsid epitopes serving simultaneously for capture and detection ofanti-capsid antibodies. A representative example is given where, in thesimultaneous assay for sandwich detection of the antigen and of theantibodies in indirect assay, for detecting the antigen, a first(capture) antibody is used, directed against the epitopes of thesequence from amino acid (AA) 100 to amino acid 130 of the HCV capsid,and a second (detection) antibody, directed against the epitopes of thesequence AA40-50, and, for detecting the antibodies, the capture antigenused contains, for its part, the sequences AA1-42 and AA66-80.

Patent application WO 01/96875 (CHIRON) describes, among others, anassay for simultaneous detection of the capsid and of the anti NS3 andNS4 antibodies, employing N-laurylsarcosine as detergent. It mentions anassay for simultaneous detection of the capsid antigen (in sandwich) andof anti-capsid and anti-non-structural HCV protein antibodies (insandwich, double-antigen). For capturing the antigen, two antibodies areused, c11-3 and c11-7, which are reputed to recognize an extensiveN-terminal portion (AA 10-53) of the HCV capsid, and for detection, athird antibody, c11-14, which is reputed to recognize a C-terminalportion (AA 120-130) of the HCV capsid. For detecting the antibodies,the capture antigen used is a fusion antigen with multiple epitopeswhich contains, fused with a fragment of superoxide dismutase (“SOD”),antigens NS3, NS4, NS5 and series of the capsid sequences from severalstrains of HCV: AA 9-53, bearing the R47L mutation, AA 64-88 and AA67-84.

Patent application EP 1 251 353 (Ortho-Clinical Diagnostics) describes a“Combo complete” assay using the same antibodies for detecting thecapsid, but without stating their origin or their epitope specificity.The anti-capsid antibodies are detected by means of a capsid antigenthat has been modified (by mutagenesis): C22KSNV47, 48 (fusion proteinwith SOD comprising the deleted capsid sequence AA 10-99 of amino acids47 and 48) or C22KSR47L (fusion protein with SOD comprising the capsidsequence AA 10-99, with a leucine replacing an arginine in position 47).

Patent application WO2003/002749 (Abbott) describes many antigens andassays for detecting HCV capsid antigen. The only “Combo complete” assaythat it describes, under the name of “Real Combo”, employs abiotinylated peptide corresponding to amino acids 11-28 of the capsid,immobilized in the solid phase, for detecting anti-capsid antibodies.For detecting the capsid, it employs the combination of Advanced LifeScience Institute antibody C11-14 (recognizing the capsid sequence AA45-50) in the solid phase and C11-10 (recognizing the capsid sequence AA32-36) labelled with acridine. Application WO 03/002 749 thereforeperforms capture of the capsid antigen and capture of the anti-capsidantibodies via two capsid sites that are clearly separate, i.e. notsuperposed (AA 11-28 for detecting the antibodies and anti-AA 32-36antibodies and AA 45-50 for detecting the antigen).

Patent application EP 1 310 512 (Ortho-Clinical Diagnostics) describesthe use of peptides containing a mutated capsid sequence, eliminatingthe possibility of binding to HCV-specific mouse monoclonal antibodies,used in a test for detecting HCV infection.

The authors of the invention described in patent applicationWO2003/095968 made certain epitopes of the target antigens used forcapturing the antibodies artificially different, by structuralmodification. The epitopes thus modified are then destroyed.Simultaneously, the antibodies used for capturing and/or detecting theantigens are for their part selected so that they recognize preciselyunmodified epitopes present on the patient's antigens, and so that theytherefore cannot bind to the modified antigens, which no longer havethese same epitopes. Since the epitopes are no longer identical, thereis no longer competition between the antibodies used for capturingand/or detecting the HCV antigen and the patient's antibodies. Patentapplication WO2003/095968 more precisely describes an HCV capsid peptidemutated in at least two separate epitope sites, and notably the peptide1-75 (G34-G44-G47).

SUMMARY OF THE INVENTION

The inventors now propose an even more advantageous method of detectingan HCV infection.

While avoiding the problem of interference, the method has excellentsensitivity, permits early detection, while allowing the patient'sserological evolution after seroconversion to be monitored.

More precisely, the invention provides a method of in-vitro detection ofinfection with a hepatitis C virus (HCV) in a biological sample,comprising the simultaneous detection of the HCV capsid protein, and ofan antibody directed against said capsid protein, said method employing,for capturing the anti-capsid antibodies, a peptide comprising anantigenic fragment derived from the truncated HCV capsid.

Thus, one object of the invention is a method of in-vitro detection ofan infection with a hepatitis C virus (HCV) in a biological sample,comprising the simultaneous detection of the HCV capsid protein, and ofan antibody directed against said capsid protein, present in thebiological sample, said method comprising a) bringing the biologicalsample in contact with a capture antibody directed against said capsidprotein, and a capture antigen capable of capturing the anti-capsidprotein antibodies present in the sample; b) incubating the mixture inconditions permitting the formation of antigen-antibody complexes; c)detecting the antigen-antibody complexes formed, which optionallyemploys a labelled detection antibody, capable of binding to thecaptured capsid protein and/or optionally also a labelled detectionantigen, capable of binding to the antibody directed against thecaptured capsid; characterized in that said capture antigen is a peptidecomprising, or consisting of, an antigenic fragment derived from the HCVcapsid from, at most, amino acid 1 to amino acid 44, said antigenicfragment comprising, or consisting of, i) a sequence from amino acids 6to 37 of the HCV capsid protein, and having at least one point mutationof at least one amino acid at positions 33 to 36, and preferably of theamino acid in position 34, or ii) a homologous sequence of the latter.

Another object of the invention is a peptide comprising said antigenicfragment.

Another object of the invention is a kit to be used for detecting aninfection with an HCV virus in a biological sample, comprising saidpeptide, as capture antigen of the anti-HCV capsid antibodies.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Here, the term “hepatitis C virus” or “HCV” covers all the strains, allthe types, subtypes and genotypes of the virus responsible for hepatitisC. The method of the invention aims in fact to detect any HCV infection,whatever its origin and its genotype. This comprises in particular thewell-known types and subtypes of the virus circulating in Europe, theUnited States, Japan, etc. (i.e. the 6 major genotypes: 1, 2, 3, 4, 5, 6and their subtypes 1a, 1b, 3a, etc.). See Stuyver et al. (1994); Bukh(1995).

The HCV capsid protein is a protein of 191 amino acids, of sequence SEQID NO:1:

1 mstnpkpqrk tkrntnrrpq dvkfpgggqi vggvyllprr gprlgvratr ktsersqprg 61rrqpipkarq pegrawaqpg ypwplygneg mgwagwllsp rgsrpswgps dprrrernlg 121kvidtltcgf adlmgyiplv gaplggaara lahgvrvled gvnyatgnlp gcsfsiflla 181llscltipas a

Unless stated otherwise, the amino acids are localized in the capsidprotein with reference to sequence SEQ ID NO:1, which is a consensussequence of genotype 1 (subtypes 1a, 1b, 1c).

Several epitopes of the capsid protein have been identified. Notably theepitopes localized between amino acid 16 and amino acid 40 are known.

The expression “from, at most, amino acid 1 to amino acid 44” means thatthe peptide with at most 44 amino acids does not extend beyond aminoacid 44 of sequence SEQ ID NO:1, but can optionally be shorter, providedit contains the sequence 6-37 (still referring to SEQ ID NO:1).

In the context of the invention, a “biological sample” preferablyconsists of a biological fluid, such as blood, plasma, serum, urine,cerebrospinal fluid, saliva, etc.

The term “antibody” refers to any whole antibody or functional fragmentof an antibody comprising or consisting of at least one antigenrecognition site, allowing said antibody to bind to at least oneantigenic determinant of an antigenic compound. As examples of antibodyfragments, we may mention the fragments Fab, Fab′, F (ab′) 2 as well asthe chains scFv (single chain variable fragment), dsFv (double-strandedvariable fragment), etc. These functional fragments can notably beobtained by genetic engineering.

The production of monoclonal antibodies or of monospecific polyclonalsera useful in the context of the invention is based on conventionaltechniques, the details of which are given later.

“Capture antibody” means an antibody or a part of an antibody,preferably fixed on a solid phase, which is capable of retaining an HCVantigen present in a biological sample, by affine binding.

The presence of antibodies and antigens in the biological sample isrevealed by specific markers. Regarding detection of the antigen, theinvention notably envisages detection by means of at least one“detection antibody”. Said labelled “detection antibody” is capable ofbinding to the captured antigen, by affine binding, recognizing anepitope site, different from that recognized by the capture antibody.Regarding detection of the antibodies, labelled anti-immunoglobulin, oranti-isotype, antibodies can notably be used, for exampleanti-immunoglobulins G in an indirect ELISA format or labelled antigensin a sandwich format.

The capture antibody and/or detection antibody recognizes the naturalepitope of portion 33-36 of the HCV capsid protein.

The term “labelled” refers both to direct labelling (via enzymes,radioisotopes, fluorochromes, luminescent compounds, etc.) and toindirect labelling (for example, via antibodies themselves labelleddirectly or by means of reagents of a labelled “affinity pair”, such as,but not exclusively, the labelled avidin-biotin pair, etc.).

“Capture antigen” means an isolated antigenic fragment, preferably fixedon a solid phase, which is capable of being recognized by anti-HCVantibodies and of permitting affine binding with the latter.

“Detection antigen” means a labelled antigen. It makes it possibleeither to detect the captured antigen by competition, or to detectantibodies by a conventional antigen-antibody-antigen sandwichtechnique, also called “double antigen sandwich” method (Maiolini et al.(1978)).

The term “specific” or “specifically”, when it refers to a recognitionor a specific binding of an antibody for an antigen, signifies that theantibody interacts with the antigen without substantial interaction withother antigens, or if we are talking of “specific” recognition with anepitope, by quasi-exclusive recognition of this epitope. Associationconstants above 10⁸ L. mol⁻¹ are preferable.

The term “homologue” refers to a peptide comprising an antigenicfragment derived from the HCV capsid of at most 44 amino acids. Thisantigenic fragment comprises a sequence differing from the sequenceranging at most from amino acids 1 to 44 of the HCV capsid protein, by aconservative substitution of one or more amino acids, or a deletion ofone or more amino acids at the N-terminal end (amino acids 1 to 5)and/or C-terminal end (amino acids 38 to 44).

Capture and Detection Antibodies and Antigens

The antibodies used in the present invention are specific antibodies ofthe antigen, and, for this reason, are monoclonal antibodies ormonospecific polyclonal antibodies, i.e. they only recognize one epitopespecifically.

The method of the invention relates more particularly to the detectionof anti-capsid antibodies, and employs capture antigens that fix theanti-capsid antibodies present in the biological sample.

The peptide intended for capturing the anti-capsid antibodies comprisesan antigenic fragment derived from the HCV capsid ranging at most fromamino acids 1 to 44, said antigenic fragment comprising a sequence fromamino acids 6 to 37 of the HCV capsid protein, or a homologous sequenceof the latter. It is therefore understood that the capture antigenaccording to the invention lacks the amino acids 45 to 191 (which arenormally present in the HCV capsid protein). According to a particularembodiment, the amino acid sequence of the peptide useful as captureantigen according to the invention comprises or consists of 32 to 44amino acids, for example consists of a sequence of 44 amino acids.

The peptide has at least one point mutation of at least one amino acidin the sequence 33-36, and preferably of the amino acid in position 34.It can be a substitution with any non-conservative amino acid. For aminoacid 34, an amino acid preferably different from an isoleucine, leucine,methionine, phenylalanine or alanine residue is used. Preferably, anuncharged amino acid is selected. Preferably, the amino acid valine inposition 34 is substituted with a glycine.

Owing to the mutation of the capture antigenic peptide, there is nointerference between the immobilized antigen-anti-capsid antibodyreaction of the sample, and the immobilized antibody-capsid antigenreaction of the sample.

According to a preferred embodiment, the capture antibody immobilized onthe solid phase is an antibody directed against the natural epitope33-36 of the capsid protein, whereas the detection antibody at c1),labelled directly or indirectly, is directed against an epitopedifferent from the natural epitopes present in portion 33 to 36, moregenerally 6 to 37, of said HCV capsid protein. For example it can be anantibody directed against epitope 44-47 of the capsid.

According to another preferred embodiment, the capture antibodyimmobilized on the solid phase is an antibody directed against anepitope different from the natural epitopes present in portion 33 to 36,more generally 6 to 37, of said HCV capsid protein—for example it can bean antibody directed against epitope 44-47 of the capsid, whereas thedetection antibody at c1), labelled directly or indirectly, is anantibody directed against the natural epitope 33-36 of the capsidprotein.

According to a particular embodiment, the capture antigen is a peptideconsisting of sequence 1 to 44 of the HCV capsid protein, and having apoint mutation of the amino acid in position 34, preferably asubstitution to glycine.

A preferred example is therefore the peptide of sequence SEQ ID NO:2.

₁MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGG G ₃₄ YLLPRRGPRL

A homologous peptide, consisting of a sequence as defined above, butadditionally truncated from 1 to 5 amino acids on the N-terminal orC-terminal side, can also be used. Thus, the peptide 6-37 can be used,having a point mutation of at least one amino acid in the sequence33-36, and preferably of the amino acid in position 34. Preferably it isthe peptide of sequence SEQ ID NO:3.

₆KPQRKTKRNTNRRPQDVKFPGGGQIVGG G ₃₄ YLL₃₇

Peptides only differing from the above peptides by conservativesubstitution are also comprised in the invention.

The expression “conservative substitution” expresses any replacement ofone amino acid residue with another, without altering the generalconformation or the antigenicity of the peptide. Conservativesubstitution includes, but is not limited to, replacement with an aminoacid having similar properties (for example shape, polarity, hydrogenbonding potential, acidity, basicity, hydrophobicity etc.). Amino acidshaving similar properties are well known by a person skilled in the art.For example, arginine, histidine and lysine are basic hydrophilic aminoacids and can be interchangeable. In the same way, isoleucine, ahydrophobic amino acid, can be replaced with a leucine, a methionine ora valine. The neutral hydrophilic amino acids that can replace oneanother include asparagine, glutamine, serine and threonine.“Substituted” and “modified” mean, according to the invention, aminoacids that have been altered or modified relative to an amino acid foundin nature.

Thus, in the context of the present invention, a conservativesubstitution is a substitution of one amino acid with another havingsimilar properties. Examples of conservative substitutions are given inTable 1 below:

TABLE 1 Conservative substitutions I Characteristics of the side chainAmino acid Non-polar G A P I L V Polar, uncharged C S T M N QPolar, charged D E K R Aromatic H F W Y other N Q D E

According to Lehninger, 1975, the conservative amino acids can also begrouped as shown in Table 2 below:

TABLE 2 Conservative substitutions II Characteristics of the side chainAmino acid Non-polar (hydrophobic) A. Aliphatic: A L I V P B. Aromatic:F W C. Containing a sulphuryl: M D. Other G Polar, unchargedA. Hydroxyl: S T Y B. Amides: N Q C. Sulphydryl: C D. Other GPositively charged (basic) K R H Negatively charged (acidic) D E

Yet another alternative conservative substitution is presented in Table3 below:

TABLE 3 Conservative substitutions III Original residue Example ofsubstitution Ala (A) Val (V), Leu (L), Ile (I) Arg (R) Lys (K), Gln (Q),Asn (N) Asn (N) Gln (Q), His (H), Lys (K), Arg (R) Asp (D) Glu (E) Cys(C) Ser (S) Gln (Q) Asn (N) Glu (E) Asp (D) His (H) Asn (N), Gln (Q),Lys (K), Arg (R) Ile (I) Leu (L), Val (V), Met (M), Ala (A), Phe (F) Leu(L) Ile (I), Val (V), Met (M), Ala (A), Phe (F) Lys (K) Arg (R), Gln(Q), Asn (N) Met (M) Leu (L), Phe (F), Ile (I) Phe (F) Leu (L), Val (V),Ile (I), Ala (A) Pro (P) Gly (G) Ser (S) Thr (T) Thr (T) Ser (S) Trp (W)Tyr (Y) Tyr (Y) Trp (W), Phe (F), Thr (T), Ser (S) Val (V) Ile (l), Leu(L), Met (M), Phe (F), Ala (A)

The peptides according to the invention can be prepared by all theclassical techniques of peptide synthesis, namely notably by chemicalsynthesis or genetic recombination. In a preferred embodiment, thepeptides are obtained by chemical synthesis. More preferably, thepeptides are obtained either by successive condensation of the aminoacid residues in the required order, or by condensation of the residueson a fragment previously formed and already containing several aminoacids in the appropriate order, or by condensation of several fragmentspreviously prepared, taking care to protect, beforehand, all thereactive functions carried by the amino acid residues, except the amineand carboxyl functions inserted in the peptide bond during condensation,and notably by Merrifield's technique of solid phase synthesis, which isadvantageous for reasons of purity, antigenic specificity, absence ofunwanted by-products and for its ease of implementation (Merrifield,(1963); R. C. Sheppard (1971); Atherton et al. (1989)). As automaticsynthesizer, it is possible to use the “9050 Plus Pep Synthesizer” fromMillipore, the “Pioneer” synthesizer from Perseptive, the “433A”synthesizer from ABI (Applied Biosystems Inc.) or the “Symphony”synthesizer from Rainin. The peptides can also be obtained byhomogeneous phase synthesis.

The antigenic peptide intended for capturing the anti-capsid antibodiescan be prolonged at its N-terminal end with a spacer.

The invention also relates to said conjugated peptide comprising theantigenic peptide, bound covalently, at its N-terminal end, to a spacer.

“Spacer” means a molecule, which is preferably a peptide, preferablyfrom 1 to 8 amino acids, preferably functionalized, i.e. having thiol,hydrazide, aldehyde functions, or a biotin, for example. It is generallya peptide of 1 to 8 amino acids, which can comprise non-natural aminoacids, such as ω-amino acids. Preferably said spacer contains at leastone cysteine residue. According to a preferred embodiment, the spacer isC-Hx, where C is a cysteine and Hx is 6-aminohexanoic acid. According toanother embodiment, the spacer is the peptide of sequence CGG.

The capture peptide can be coupled covalently to itself or it can becoupled to a carrying molecule via the spacer. The spacer thus makes itpossible to optimize the attachment of the antigenic peptide on a solidsurface, a protein or a soluble functionalized polymer and/or makes itpossible to conjugate several antigenic molecules. Preferably, thecarrying molecule is a protein such as bovine serum albumin (BSA), orovalbumin. According to another embodiment, the peptide is bound, viathe spacer, to an amino-dextran.

Thus, according to a particular embodiment, the peptide conjugatecomprises a spacer bound covalently to several antigenic peptides, whichmay be identical or different. Preferably a homodimer is thus formed.

Especially preferably, the capture antigen of the anti-capsid antibodiesis only one antigenic peptide as defined above (for example the peptideof sequence SEQ ID NO:2).

However, in a particular embodiment, the capture antigen according tothe invention, as defined above (for example the peptide of sequence SEQID NO:2), can be used in combination with another capture antigen(preferably present on the same solid phase as the capture antigenaccording to the invention), whose amino acid sequence comprises orconsists of a fragment of the HCV capsid protein, ranging from aminoacids 45 or 46 to one of the amino acids between 60 and 80, preferablybetween 65 and 75 or between 68 and 75 (still referring to SEQ ID NO:1).In a more particular embodiment, the additional capture antigen consistsof the sequence 45-65, 45-66, 45-67, 45-68, 45-69, 45-70, 45-71, 45-72,45-73, 45-74, or 45-75, or the sequence 46-65, 46-66, 46-67, 46-68,46-69, 46-70, 46-71, 46-72, 46-73, 46-74, or 46-75. As examples, we maymention the peptide 45-65, the peptide 45-68 or, more preferably, thepeptide 45-75.

Moreover, detection of the anti-capsid antibodies can be combined withthe detection of antibodies to another HCV protein. Various capture, ordetection, antigens can be combined together. This embodiment, whichemploys several different capture, and/or detection, antigens, allowsfor example the simultaneous detection of anti-capsid antibodies and ofanti-non-structural protein antibodies, for example NS3 and/or NS4 orNS5 of HCV. Simultaneous detection of anti-capsid antibodies and ofanti-NS3 antibodies is particularly preferred. The invention alsocomprises simultaneous detection of capsid antigen, of anti-capsidantibody, of anti-envelope structural proteins E1 and/or E2 antibodies,of envelope E1 and/or E2 antigen, and/or of anti-non-structural proteinsNS3 and/or NS4 or NS5 of HCV antibodies. Other conceivable combinationsalso form part of the invention.

Methods of Detection

The method of the invention comprises simultaneous detection of the HCVcapsid protein, and of an antibody directed against said capsid protein,present in the biological sample, said method comprising a) bringing thebiological sample in contact with a capture antibody directed againstsaid capsid protein, and a capture antigen capable of capturinganti-capsid protein antibodies present in the sample; b) incubating themixture in conditions permitting formation of antigen-antibodycomplexes; c) detecting the antigen-antibody complexes formed, whichoptionally employs a labelled detection antibody, capable of binding tothe captured capsid protein and/or optionally also a labelled detectionantigen, capable of binding to the antibody directed against thecaptured capsid.

In general, and unless stated otherwise, the capture antigen refers hereboth to the antigenic fragment derived from the HCV capsid from, atmost, amino acid 1 to amino acid 44, and to a conjugated peptidecomprising said fragment, as described above.

The biological sample can optionally be treated in a preliminary step,or can be brought in contact with the capture antigen and the captureantibody in conditions promoting exposure of the antigens to bedetected.

Advantageously, the sample is treated with a denaturing agent, beforedetection, and preferably before it is brought in contact with theantibodies used. This denaturing agent can notably consist of one ormore detergents of the non-ionic type, such as, for example, Tween 20,Triton X-100, Nonidet P-40 (NP40) (tert-octylphenoxy poly(oxyethylene)ethanol, also called IGEPAL CA630), n-octyl beta-D-glucopyranoside, oran acidic solution.

This combined immunoassay can be performed according to various formatsthat are well known by a person skilled in the art: in solid phase or inhomogeneous phase; once or twice; in a double sandwich method (sandwichfor both detections of antigens and of antibodies); or in an indirectmethod (for detecting antibodies) combined with a sandwich method (fordetecting antigen), as non-limiting examples.

According to a preferred embodiment, the capture antibody and thecapture antigen are immobilized on a solid phase. As non-limitingexamples of a solid phase, it is possible to use microplates, inparticular polystyrene microplates, such as those marketed by thecompany Nunc, Denmark. It is also possible to use solid particles orbeads, paramagnetic beads, such as those supplied by Dynal orMerck-Eurolab (France) (under the brand name Estapor), or test tubes ofpolystyrene or polypropylene, or a nitrocellulose membrane, etc.

An immunoassay format of the sandwich type between two antibodies (forcapture and for detection) is particularly advantageous for detectingthe antigens present in the biological sample, whereas the antibodiescan be detected by employing a capture antigen and a labelled conjugate,which is fixed on the antibody (according to a format commonly called“indirect format”), for example the labelled protein A or a labelledanti-immunoglobulin, or anti-isotype, antibody. It is also possible todetect the antibodies advantageously by employing a capture antigen anda labelled antigen, which are fixed on the antibody (according to aformat designated as “antigen-antibody-antigen sandwich” or “doubleantigen sandwich”).

An immunoassay format for detecting the antigens by competition is alsopossible. Other types of immunoassay can also be envisaged and are wellknown by a person skilled in the art.

The simultaneous detection of the HCV antigen and of the anti-HCVantibodies according to the invention can be performed once, namely bysimultaneously bringing in contact the biological sample, the detectingmeans, such as notably the detection antibody or antibodies, at the sametime as the capture antibody or antibodies and the capture antigen orantigens. In this case, the immunoassay for detecting the antigen andthe immunoassay for detecting the antibodies are both preferablyperformed in a sandwich. Alternatively, the detecting means, such asnotably the detection antibody or antibodies, can be added to themixture secondly, i.e. after the first antigen-antibody complexes haveformed. This is then called a two-step assay.

According to a preferred embodiment of the invention, the method ofdetecting infection with the hepatitis C virus (HCV) in a biologicalsample comprises: a) bringing the sample in contact with a captureantibody of the HCV capsid protein and a capture antigen of the anti-HCVcapsid antibodies fixed on a solid phase; b) incubating the mixture inconditions permitting formation of antigen-antibody complexes; c)separating the solid phase and the liquid phase; d) bringing the solidphase in contact with a labelled detection antibody capable of bindingthe captured HCV antigen, and one or more labelled anti-immunoglobulin,or anti-isotype, antibodies, capable of binding the captured anti-HCVantibody.

ELISA assays, radioimmunoassays, or any other detection technique can beemployed for revealing the presence of the antigen-antibody complexesformed.

Detection of the presence of antigens or of antibodies in the biologicalsample can be supplemented with quantification, for example bymeasurement of the signals emitted by the markers (colour, luminescence,radioactivity, etc.), according to the standard techniques familiar to aperson skilled in the art.

Kits

Kits and reagents useful for detecting an HCV infection in a biologicalsample, according to the method of the invention, can be supplied forsimple practical application of the invention that is applicable tonumerous biological samples.

The invention therefore provides a kit that can be used for detecting aninfection by an HCV virus in a biological sample, comprising anantigenic peptide capable of capturing the anti-HCV capsid antibodies.The kit can further comprise means for detecting said anti-HCVantibodies present in the biological sample and complexed to saidcapture antigen, said detecting means preferably being a labelledanti-immunoglobulin or anti-isotype antibody.

Advantageously, said kit can contain several antigens and severalcapture antibodies.

The kit can in addition contain antigens of other proteins of the virus,such as NS3 and/or NS4, and/or NS5, these antigens then being intendedto capture anti-NS3 and/or anti-NS4, and/or NS5 antibodies. Preferablythese antigens are mixed with the capture antigen derived from thecapsid.

As described above, the capture antibody and the capture antigen can bepresented advantageously in immobilized form on a solid phase, such as amicroplate.

A preferred kit comprises

a1) a capture antigen, which is a peptide comprising, or consisting of,an antigenic fragment derived from the HCV capsid from, at most, aminoacid 1 to amino acid 44, said antigenic fragment comprising, orconsisting of, i) a sequence from amino acids 6 to 37 of the HCV capsidprotein, and having at least one point mutation of the amino acid inposition 34, or ii) a homologous sequence of the latter;

a2) preferably also capture antigens of the anti-non-structural proteinantibodies comprising some or all of the non-structural proteins NS3,NS4, and/or NS5,

b) a capture antibody directed against the HCV capsid protein;

said capture antigen and said capture antibody being immobilized on asolid phase; and

c1) a labelled detection antibody,

c2) an anti-immunoglobulin antibody or antibodies or optionally alabelled detection antigen, which comprises, or is, an antigenicfragment that is a peptide comprising, or consisting of, an antigenicfragment derived from the HCV capsid from, at most, amino acid 1 toamino acid 44, said antigenic fragment comprising, or consisting of, i)a sequence from amino acids 6 to 37 of the HCV capsid protein, andhaving a point mutation of at least one amino acid at positions 33 to 36or ii) a homologous sequence of the latter.

The capture antibody and/or detection antibody recognizes the naturalepitope of portion 33-36 of the HCV capsid protein.

According to a preferred embodiment, the capture antibody immobilized onthe solid phase is an antibody directed against the natural epitope33-36 of the capsid protein, whereas the detection antibody at c1),labelled directly or indirectly, is directed against an epitopedifferent from the natural epitopes present in portion 33 to 36, moregenerally 6 to 37, of said HCV capsid protein. For example it can be anantibody directed against epitope 44-47 of the capsid.

According to another preferred embodiment, the capture antibodyimmobilized on the solid phase is an antibody directed against anepitope different from the natural epitopes present in portion 33 to 36,more generally 6 to 37, of said HCV capsid protein—for example it can bean antibody directed against epitope 44-47 of the capsid, whereas thedetection antibody at c1), labelled directly or indirectly, is anantibody directed against the natural epitope 33-36 of the capsidprotein.

The kit can further comprise a detergent, more particularly a non-ionicdetergent known by a person skilled in the art.

The following examples illustrate the invention without limiting itsscope.

EXAMPLES Detection of an Infection with the Hepatitis C Virus

Materials for the Assay Protocol:

-   -   1) Solid phase selected: Maxisorp microplate, Nunc (Denmark).    -   2) Anti-capsid monoclonal antibody-biotin conjugate (“acm-POD”):        a conjugate of the anti-capsid monoclonal antibody, Acm 2        labelled with peroxidase is prepared according to the protocol        described in patent application EP 0 752 102.    -   Another anti-capsid monoclonal antibody, Acm 1 is also used.    -   3) Mouse anti-IgG (Fc_(γ)) human polyclonal antibody-POD        conjugate: this conjugate is obtained from Jackson        lmmunoresearch Laboratories, USA (indirect ELISA format).        Biotin-labelled capsid peptide conjugate (sandwich format).    -   4) Diluents for the 1st and 2nd steps of the protocols according        to the invention:    -   Diluent for the 1st step: Tris buffer, NaCl 0.05M, at pH 6.7        with addition of IGEPAL CA 630 (Sigma) at 0.25%.    -   Diluent for the 2nd step: citrate buffer (50 mM), at pH 6.7,        solution in glycerol at 20%.    -   5) Development solution: the development solution was composed        of    -   5a) a substrate buffer: solution of citric acid (0.075M), and of        sodium acetate (0.1M), at pH 4.0, containing H₂O₂ at 0.015% and        dimethylsulphoxide (DMSO) (PROLABO) at 4%, and    -   5b) a chromogen: solution containing tetramethylbenzidine (TMB)        (8 mM), (Sigma).

Methods:

Protocol for Simultaneous Detection of the Capsid Antigen and of theAntibodies (Anti-Capsid and Anti NS3, NS4) of the Hepatitis C Virus in aSample (Serum or Plasma)

The principle of the assay is based on an immunoenzyme method of thesandwich type for detecting the antigen, and of the indirect type, fordetecting the antibodies.

It is based on the following steps:

A sensitizing solution is first prepared:

-   -   with a mixture of HCV antigens: a mutated peptide CHx-1-44 (G34)        (capsid) comprising the sequence SEQ ID No.2 and two recombinant        proteins produced by Escherichia coli from clones selected from        the non-structural regions NS3 (AA 1192-1657), and NS4 (AA        1694-1735) and    -   with an anti-capsid monoclonal antibody (Acm 1), in buffer Tris        0.5M, pH 7.4.

The wells of a microtitre plate (Nunc, Maxisorp) are then sensitizedwith the above solution at a rate of 110 μl per well. The microtitreplates are incubated overnight at room temperature (18-24° C.).

After removing the sensitizing solution, the plates are washed withphosphate buffer (0.01M, pH 7.4) containing 0.1% of Tween 20, thensaturated by adding a phosphate buffer (0.01M, pH 7) containing 5% ofsucrose, 25% of skimmed milk (Candia™, France, or any other equivalentcommercial skimmed milk) and 10 mM of EDTA. 100 μl of diluent for the1st step, containing the peroxidase-labelled anti-capsid monoclonalantibody Acm 2, then 50 μl of the sample (serum or plasma), aredistributed successively in each well.

The reaction mixture is incubated at 37° C. for 1.5 h. Any HCV capsidantigens that may be present become attached to the solid-phasemonoclonal antibody Acm 1 and form complexes with theperoxidase-labelled anti-capsid monoclonal antibody Acm 2. Moreover, ifanti-HCV antibodies are present, they bind to the antigens fixed on thesolid phase.

The plates are then washed (3 times) with a washing solution (bufferTris NaCl 0.01 M, pH 7.4 with addition of Tween 20 at 0.1%).

100 μl of diluent for the 2nd step containing peroxidase-labelledanti-human IgG antibodies or the biotin-labelled capsid peptide isdistributed in each well. The reaction mixture is incubated at roomtemperature (18-24° C.) for 30 minutes. The labelled anti-human IgGantibodies or the biotin-labelled capsid peptide in their turn becomefixed to the specific antibodies retained on the solid phase.

The plates are then washed (5 times) with a washing solution (bufferTris NaCl 0.01 M, pH 7.4 with addition of Tween 20 at 0.1%). The unboundanti-human IgG conjugate is thus removed.

100 μl of a development solution (substrate buffer+chromogen) isdistributed in each well. The reaction is allowed to develop in the darkfor 30 minutes at room temperature (18-24° C.).

Then 100 μl of stopping solution (1N H₂SO₄) is distributed in each well.

After stopping the reaction, the optical density is read on aspectrophotometer at 450/620 nm.

Definition of the Threshold Value:

The threshold value was determined after statistical analysis of thedata for specificity and sensitivity using the ROC (Receiver OperatingCharacteristic) curve (Berck and Schultz, (1986)).

The specificity study was based on 5000 samples from healthy subjectsand the sensitivity study was based on HCV positive samples (notablycommencement of seroconversion) from commercial panels: BBI (BostonBiomedica Company, USA), Impath (USA), Serologicals (USA), Nabi (USA),ProMedDx (USA)).

The threshold value is calculated for each plate from the signalobtained on a positive control, divided by a constant coefficient X,specific to the test. It is approx. 0.45 unit of OD (optical density) inthe example presented in indirect format.

Example 1 Detection of Anti-Capsid Antibodies in Samples (Sera orPlasmas) that are Negative for HCV Antigen

Three panels of seroconversion samples PHV 905, 907 and 914 (i.e. twoconsecutive samples of sera or plasmas, taken from two patients afterHCV infection or in the course of seroconversion) that are commerciallyavailable (Seracare Life Sciences/BBI Diagnostics, USA) were testedaccording to the sandwich protocol. The capsid peptide 1-44 G34 (SEQ IDNO:2) was then labelled with biotin and the complex was revealed withstreptavidin-peroxidase. It is therefore a single anti-capsid detection.These samples were found to be positive with a threshold valuecalculated from the mean value of three negative samples plus 6 standarddeviations (OD: 0.077).

TABLE 1 Sample Optical density 905-08 0.058 negative 905-09 0.209positive 907-05 0.480 positive 907-06 0.694 positive 914-08 0.409positive 914-09 0.824 positive Neg 1 0.043 negative Neg 2 0.048 negativeNeg 3 0.044 negative

The invention therefore makes it possible to detect, in subjectscontaminated with HCV, samples that are positive for anti-capsidantibodies.

Example 2 Comparison of Performance and Classification of VariousTechniques on Seroconversion Sera that are Positive for Antibody and/orAntigen

9 commercially-available seroconversion panels (Seracare LifeSciences/BBI, USA; Impath, USA), having different specificities, weretested with the assay according to the invention following the protocolindicated, and the results were compared with a series ofcommercially-available tests. This comparison takes into account thenumber of days of delay in detection relative to RNA detection (PCRassay). Thus, the kit having the smallest sum gives the best performancefor early detection. These panels are found to be positive for antigenand/or antibody directed against the NS3 proteins and capsid of the HCVvirus (characterization carried out by the RIBA test marketed by thecompany Ortho Clinical Diagnostics).

TABLE 2 Number of days of delay of detection relative to detection byPCR “Ortho HCV 3.0 short Panel Axsym HCV incubation” Impath/ Inventionversion3 (Ortho Clinical Access HCV Ab Target BBI protocol (Abbott)Diagnostics) Plus (BIO-RAD) antigen  912 0 7 7 7 Capsid  913 0 12 7 7Capsid 6215 0 20 20 20 Capsid Sub-total 0 39 34 34  915 5 5 14 17 NS36212 12 12 12 23 NS3 6224 11 19 19 19 NS3 9041 27 62 62 62 NS3 9044 0 2125 25 NS3 9047 0 28 28 28 NS3 Sub-total 55 147 160 174 Total 60 186 194208

The invention makes it possible to detect samples positive for antibodyand antigen, earlier than an antibody assay.

References

-   -   Atherton et al. (1989) “Solid phase peptide synthesis, a        practical approach”, IRL Press, Oxford University Press, pp.        25-34    -   Bukh, Semin. Liver Dis. (1995) 15: 41-63;    -   Maiolini et al., (1978) Journal of Immunological Methods, 20,        pp. 25-34    -   Merrifield (1963) J. Amer. Chem. Soc, 85, pp. 2149-2154    -   Sheppard, in “Peptides 1971”, Nesvadba H (ed.) North Holland,        Amsterdam, p. 111    -   Stuyver et al. (1994), P.N.A.S. USA, 91, pp. 10134-10138

1-20. (canceled)
 21. A method of in-vitro detection of an infection witha hepatitis C virus (HCV) in a biological sample, comprising thesimultaneous detection of the HCV capsid protein, and of an antibodydirected against said capsid protein, present in the biological sample,said method comprising a) bringing the biological sample in contact witha capture antibody directed against said capsid protein, and a captureantigen capable of capturing anti-capsid protein antibodies present inthe sample; b) incubating the mixture in conditions permitting formationof antigen-antibody complexes; c) detecting the antigen-antibodycomplexes formed, which optionally employs a labelled detectionantibody, capable of binding to the captured capsid protein and/oroptionally also a labelled detection antigen, capable of binding to thecaptured anti-capsid antibody; wherein said capture antigen is a peptidecomprising, or consisting of, an antigenic fragment derived from the HCVcapsid from, at most, amino acid 1 to amino acid 44, said antigenicfragment comprising, or consisting of, i) a sequence from amino acids 6to 37 of the HCV capsid protein, and having a point mutation of at leastone amino acid at positions 33 to 36, or ii) a homologous sequence ofthe latter.
 22. The method according to claim 21, wherein the captureantigen is a peptide having a point mutation of amino acid
 34. 23. Themethod according to claim 22, wherein the capture antigen is a peptideconsisting of amino acids 1 to 44 of the HCV capsid protein, and havinga point mutation of the amino acid in position
 34. 24. The methodaccording to claim 21, wherein the amino acid in position 34 is aglycine residue.
 25. The method according to claim 21, wherein, saidcapture antibody and said capture antigen are immobilized on a solidphase.
 26. The method according to claim 25, wherein the solid phasefurther comprises at least one antigen derived from an HCV protein whichis not the capsid.
 27. The method according to claim 21, wherein thedetection antibody is added to the mixture after the antigen-antibodycomplexes have formed.
 28. The method according to claim 21, wherein thedetection antibody, and/or the detection antigen, is brought in contactwith the biological sample at the same time as the capture antibody andthe capture antigen.
 29. An isolated peptide comprising, or consistingof, an antigenic fragment derived from the HCV capsid from, at most,amino acid 1 to amino acid 44, said antigenic fragment comprising, orconsisting of, a sequence from amino acids 6 to 37 of the HCV capsidprotein, and having a point mutation of at least one amino acid atpositions 33 to 36, or a homologous sequence of the latter.
 30. Thepeptide according to claim 29, said peptide having a point mutation ofamino acid
 34. 31. The peptide according to claim 30, said peptideconsisting of amino acids 1 to 44 of the HCV capsid protein and having apoint mutation of the amino acid in position
 34. 32. The peptideaccording to claim 29, wherein the amino acid in position 34 is aglycine residue.
 33. A conjugated peptide comprising the peptide ofclaim 29 bound covalently, at its N-terminal end, to a spacer.
 34. Theconjugated peptide according to claim 33, wherein the spacer is C-Hx,where C is a cysteine and Hx is 6-aminohexanoic acid.
 35. The conjugatedpeptide according to claim 33, wherein the peptide is coupled covalentlyto itself or has a carrying molecule via the spacer.
 36. A kit fordetecting an infection with an HCV virus in a biological samplecomprising a peptide according to claim 29 as a capture antigen ofanti-HCV capsid antibodies.
 37. The kit according to claim 36 furthercomprising a means for detecting said anti-HCV antibodies present in thebiological sample and complexed to said capture antigen, said detectingmeans being a labelled anti-immunoglobulin antibody or anti-isotypeantibody.
 38. The kit according to claim 36, said kit comprising: a1) acapture antigen for anti-HCV capsid antibodies; a2) capture antigens foranti-non-structural protein antibodies selected from non-structuralproteins NS3, NS4, and/or NS5, b) a capture antibody directed againstthe HCV capsid protein; said capture antigen and said capture antibodybeing immobilized on a solid phase; and c1) a labelled detectionantibody, and c2) an anti-immunoglobulin antibody or antibodies oroptionally a labelled detection antigen.
 39. The kit according to claim36, said kit comprising a capture antibody for anti-HCV capsidantibodies, immobilized on the solid phase, directed against the naturalepitope 33-36 of the capsid protein, and a detection antibody labelleddirectly or indirectly, which is directed against an epitope differentfrom the natural epitopes present in portion 33 to 36 of said HCV capsidprotein.
 40. The kit according to claim 36, said kit comprising acapture antibody of the anti-HCV capsid antibodies, immobilized on thesolid phase, directed against an epitope different from the naturalepitopes present in amino acids 33 to 36 of said HCV capsid protein, anda detection antibody labelled directly or indirectly, directed againstthe natural epitope present in amino acids 33-36 of said HCV capsidprotein.